Volumetric imaging of erythrocytes using label‐free multiphoton photoacoustic microscopy

Abstract

Photoacoustic microscopy (PAM) is an imaging modality well suited to mapping vasculature and other strong absorbers in tissue. However, one of the primary drawbacks to PAM when used for high-resolution imaging is the relatively poor axial resolution due to the inverse dependence on the transducer bandwidth. While submicron lateral resolution PAM can be achieved by tightly focusing the excitation light, the axial resolution is fundamentally limited to 10s of microns for typical transducer frequencies. Here we present a multiphoton PAM technique called transient absorption ultrasonic microscopy (TAUM), which results in a completely optically resolved voxel with an experimentally measured axial resolution of 1.5 microns. This technique is demonstrated by imaging individual red blood cells in three dimensions in blood smear and ex vivo tissues. To the best of our knowledge, this is the first demonstration of fully resolved, volumetric photoacoustic imaging of erythrocytes.

Emerging concepts in functional and molecular photoacoustic imaging

“…This nonlinear dependence offers PAM the optical-sectioning capability equivalent to that of two-photon microscopy. Indeed, with this pump-probe strategy, the axial resolution of PAM has been refined down to 1.5 μm—approaching the lateral resolution defined by optical focusing [31]. The isotropic resolution enables three-dimensional visualization of the biconcave shape of a single RBC (Fig.…”

Photoacoustic microscopy of single cells employing an intensity-modulated diode laser

“…It is well known that hemoglobin provides strong optical absorption in the visible and gives an excellent photoacoustic contrast. Various groups have used this high endogenous contrast for demonstrating photoacoustic imaging of single red blood cells using time-domain PAM [4][5][6][7][8] . Frequency-domain PAM of red blood cells has been demonstrated by Winkler et al 9 and Langer et al 3 .…”

Multiview optical resolution photoacoustic microscopy

“…However, these methods require custom designed hardware, and suffer from the severe attenuation of high frequency ultrasonic waves in biological tissue and coupling media, limiting the imaging depth and the working distance. Meanwhile, optical sectioning has been achieved in OR-PAM through photoacoustic nonlinearity [12,13]. However, in these optically-sectioned PAM techniques, because depth scanning is required to acquire A-line signals, the imaging speed is reduced.…”